Nuclear pore complexes (NPCs) mediate the bidirectional transport of proteins, RNAs and ribonucleoprotein complexes across the double-membrane nuclear envelope of eukaryotic cells. Consequently, proper NPC function is essential for a wide variety of cellular biosynthetic and regulatory processes. Altered structural and functional properties of the NPC are linked with various human diseases including leukemias, cancers and primary biliary cirrhosis. The molecular trafficking through NPCs is highly regulated and delicately balanced; inefficient or excess transport of a single gene regulatory factor that shuttles between cytoplasmic and nucleoplasmic compartments, such as a tumor suppressor, is associated with various cancers. Alzheimer's and Huntington's disease may also be linked to nuclear transport. While many protein components of the NPC itself and many soluble protein cofactors have been identified and extensively studied, the molecular mechanisms of pore selectivity and of cargo passage through the NPC remain largely unknown. To further examine the fundamental characteristics of nucleocytoplasmic transport, single molecule fluorescence (SMF) microscopy and single particle tracking techniques were developed to directly observe molecules trafficking through NPCs with up to 1 ms time resolution. This approach allows direct measurement of cargo translocation times and their import efficiencies, and allows characterization of various aspects of cargo movement within the NPC. Surprisingly, the Vmax for transport can be altered at least ~10-fold by changing the importin 2 concentration in vitro. It remains unclear the extent to which cells utilizes this mechanism to actively regulate nuclear trafficking rates in response to need. The goals of the proposed research are to fundamentally advance our knowledge of NPC function via SMF microscopy. The Specific Aims of the project are: (1) to characterize Imp ?/CAS complex assembly during nuclear import and disassembly during nuclear export; (2) to determine the effect of transport pathway overlap on the translocation time and import efficiency of signal-dependent and -independent cargos; (3) to determine the number of Imp ? cofactors in NPCs at steady-state in vivo and as-isolated in permeabilized cells; and (4) to determine the effects of the number of nuclear localization sequences on a cargo's interaction frequency, translocation time, import efficiency and average distribution within the FG-Nup network. These experiments are designed to explore the wide parameter space enjoyed by NPCs as they transport a variety of cargos by distinct pathways, with the expectation that they will fundamentally advance our understanding of various mechanisms of nucleocytoplasmic transport. Public Health Relevance: Since nuclear pore complexes (NPCs) provide a focal point for the relay of essential materials and information between the cytoplasm and nucleus, dysfunction of the nucleocytoplasmic transport system has grave consequences for the health and viability of the cell. For example, NPC structure and function has been linked to leukemias, cancers and primary biliary cirrhosis, and possibly to Alzheimer's and Huntington's diseases. The basic biochemical mechanisms of nucleocytoplasmic transport will be characterized so that future investigations can be founded on a firm understanding of how transport maintains, or through dysfunction fails to maintain, metabolic regulation and organization in cells and tissues.